Both neural and weight-bearing activities have been shown to be important physiological factors regulating slow skeletal muscle phenotype in the adult animal. While these phenotypic changes have been well characterized, there is not much known about the molecular mechanisms through which neuromuscular activity regulates the slow contractile protein isoform genes. The long-term objectives of this project are to understand mechanisms through which contractile protein gene families are regulated in the acquisition and maintenance of diverse skeletal muscle fiber types. Previous work from this lab has shown that - 270 bp of the myosin light chain 2 slow (MLC2slow) promoter is sufficient to direct both slow nerve and mechanical load dependent expression. Further analyses have identified that the CACC and MEF2 sites within this 270 bp region cooperate in regulating transcription in response to slow innervation. The specific aims of this proposal are: 1) to identify the proteins that contribute to nerve and mechanical load dependent transcription factor complex formation at the CACC and MEF2 sites of the MLC2slow promoter; 2) to determine mechanisms by which neural and mechanical activity regulate the transcriptional activity of the factors identified in Aim 1; and 3) To determine the mechanisms by which altered cellular calcium modulates transcription factor functions in vitro and in vivo. This proposal combines both in vitro and in vivo approaches to identify the critical factors and pathways responsible for physiological regulation of the MLC2slow promoter. This work has broad application to basic and applied areas of biomedical and health science fields. At the basic science level, new insight will be gained into mechanisms by which DNA elements and factors regulate a specific contractile protein isoform gene. Contractile proteins are a major topic of investigation not only for muscle biology, but their function and regulation are also implicated in maintenance of cell architecture and morphology, cell cycle events, and cell transformation/cancer. At the more applied level, because of the common occurrence of muscle regeneration in humans, this study will have wide clinical application. Muscle regeneration does occur following muscle transplant surgery for correction of facial paralysis; following muscle damage due to mechanical, thermal, or metabolic stress; as well as its association with dystrophic muscle pathologies.